With the emergence of chemical biology, the use of pharmacological inhibitors in biological research has been expanding. SP600125 is a low-molecular weight compound that has been widely used to inhibit c-Jun-N-terminal kinase (JNK). A recent publication by Tanemura et al. (J. Biochem. 145:345-354, 2009) indicated that SP600125 also inhibits phosphatidylinositol 3-kinase (PI3K) in an isoform-selective fashion: it efficiently inhibited the delta isoform of p110 catalytic subunit (p110δ), which is primarily expressed in leucocytes, but neither of the ubiquitously expressed isoforms, p110α and p110γ. Here, I discuss what we learn from such unpredicted off-target effects of pharmacological inhibitors.